The major aim of this research is to determine the functional significance of specific nucleotides in transfer RNA and the role of tRNA in normal and abnormal mammalian cellular development. We have recently reported that a unique class of tRNA in wheat and beef lacks ribothymidine. These tRNAs are all acceptable substrates for E. coli uracil (ribothymidine) forming methylase. Ribothymidine is generally considered to be a universal nucleotide in tRNA since it is present at position 23 from the 3' end in the over 50 tRNAs whose sequences are known, except for some tRNAs from Mycoplasma, tRNAs involved only in peptioglycan synthesis, and tRNAs involved only in chain initiation. Therefore, this universal nucleotide may be required for protein synthesis in higher organisms. The nucleotide sequence of wheat embryo tRNA Gly has been determined and was shown to completely lack ribothymidine in its normal position; instead it contained uridine. I propose to study this unique class of tRNA in beef liver by isolating the tRNAs which are acceptable substrates for the E. coli uracil methylase, identifying them and sequencing these tRNAs. Thus, I will determine not only which tRNAs in beef lack ribothymidine but also what unique structural parameters exist for this unique class of tRNA. Furthermore, I will attempt to determine the functional significance of ribothymidine by comparing the reaction of methylated and unmethylated wheat tRNA Gly, in a wheat embryo in vitro protein synthesizing system, using viral RNA. In addition, this class of tRNA will be investigated in other beef organs (such as brain), in fetal calf liver, and in human placental tRNA. The long range goal of this research is to understand the function of specific nucleotides in tRNA and to apply this knowledge to determine the role of altered tRNAs in both normal and abnormal cellular development.